Following de novo fatty acid biosynthesis in the chloroplasts of higher plants (Ohlrogge and Browse, Plant Cell 7: 957-970, 1995), the majority of plastid-synthesized fatty acids are exported as palmitoyl-CoA and oleoyl-CoA to the endoplasmic reticulum (ER) for glycerolipid biosynthesis (Browse et al., Biochem J 235: 25-31, 1986; Maréchal et al., Physiol Plant 100: 65-77, 1997). Although these acyl-CoA derivatives move between the plastids and the ER via the cytosol (Ohlrogge and Browse, Plant Cell 7: 957-970, 1995), proteins that facilitate such transfer have not been identified. A potential candidate is the 10-kD acyl-CoA-binding protein (ACBP) in Arabidopsis thaliana because its derivative has been shown to bind oleoyl-CoA and protect it from degradation by microsomal acyl hydrolases (Engeseth et al., Arch Biochem Biophys 331: 55-62, 1996). To experimentally verify its subcellular localization and biological functions, we carried out further investigations on the 10-kD Arabidopsis ACBP.
In Arabidopsis, a total of six forms of acyl-CoA binding proteins (ACBPs) are present and they include the 10-kD ACBP (Engeseth et al., Arch Biochem Biophys 331: 55-62, 1996) hereby designated as ACBP6 (Xiao et al., Plant J 54: 141-151, 2008), and five other forms ranging from 37.5 to 73.1 kD (Leung et al., Plant Mol Biol 55: 297-309, 2004). Membrane-associated ACBP1 and ACBP2 are subcellularly localized to the ER and plasma membrane (Chye et al., Plant J 18: 205-214, 1999; Li and Chye, Plant Mol Biol 51: 483-492, 2003), ACBP3 is extracellularly-targeted (Leung et al., Planta 223: 871-881, 2006) and kelch-motif-containing ACBP4 and ACBP5 (Leung et al., Plant Mol Biol 55: 297-309, 2004), as well as ACBP6 are localized in the cytosol (Chen et al., Plant Physiol 148: 304-315). Only homologs of ACBP6 have been well-characterized in other eukaryotes (Hills et al., Plant Mol Biol 25: 917-920, 1994; Faergeman and Knudsen, Biochem J 323: 1-12, 1997). Domains that potentially mediate protein-protein interactions, ankyrin repeats (ACBP1 and ACBP2) and kelch motifs (ACBP4 and ACBP5) (Leung et al., Plant Mol Biol 55: 297-309, 2004; Li and Chye, Plant Mol Biol 54: 233-243, 2004), are evident in the larger ACBPs. Using His-tagged recombinant proteins and site-directed mutagenesis, the function of the acyl-CoA-binding domain in binding acyl-CoA esters was established for ACBP1 to ACBP5 (Chye et al., Plant Mol Biol 44: 711-721, 2000; Leung et al., Plant Mol Biol 55: 297-309, 2004; Planta 223: 871-881, 2006). Differential binding to various acyl-CoA esters imply that Arabidopsis ACBPs have various cellular functions.
Mammalian homologs of ACBP6 bind and transport cytosolic acyl-CoA esters and participate in gene regulation by interacting with nuclear factor-4α, a transcriptional activator of genes associated with lipid and glucose metabolism in nuclei of rat hepatocytes (Mikkelsen and Knudsen, Biochem J 248: 709-714, 1987; Black et al., J Nutr 130: 305S-309S, 2000; Elholm et al., J Lipid Res 41: 538-545, 2000; Petrescu et al., J Biol Chem 278: 51813-51824, 2003). The 10-kD bovine ACBP has been identified as a cytosolic protein (Mikkelsen and Knudsen, Biochem J 248: 709-714, 1987) while ACBP6 homologs are localized in the cytoplasm and nuclei of both monkey kidney fibroblast CV-1 cells (Helledie et al., J Lipid Res 41: 1740-1751, 2000) and human hepatocellular liver carcinoma cells (Nitz et al., Int J Biochem Cell Biol 37: 2395-2405, 2005).
ACBP6 homologs have been identified in phloem exudates in cucumber (Cucumis sativus), pumpkin (Cucurbita maxima; Walz et al., Photochemistry 65: 1795-1804, 2004) and rice (Oryza sativa) (Suzui et al., J Exp Bot 57: 2571-2576, 2006), suggesting that plant 10-kD ACBPs may be associated with long-distance transport (possibly of long-chain acyl-CoA esters) and/or in stress and defense since phloem proteins primarily belong to these classes (Walz et al., Photochemistry 65: 1795-1804, 2004; Suzui et al., J Exp Bot 57: 2571-2576, 2006). Our investigations on the response of Arabidopsis ACBP6 to abiotic and biotic stresses further showed that ACBP6 expression is cold (4° C.)-inducible, the acbp6 knockout mutant displays enhanced sensitivity to freezing treatment (−8° C.), and transgenic Arabidopsis overexpressing ACBP6 are conferred freezing-tolerance (Chen et al., Plant Physiol 148: 304-315).